Protein biopolymer markers predictive of insulin resistance

ABSTRACT

The instant invention involves the use of a combination of preparatory steps in conjunction with mass spectroscopy and time-of-flight detection procedures to maximize the diversity of biopolymers which are verifiable within a particular sample. The cohort of biopolymers verified within such a sample is then viewed with reference to their ability to evidence at least one particular disease state; thereby enabling a diagnostician to gain the ability to characterize either the presence or absence of said at least one disease state relative to recognition of the presence and/or the absence of said biopolymer, predict disease risk assessment, and develop therapeutic avenues against said disease.

FIELD OF THE INVENTION

[0001] This invention relates to the field of characterizing theexistence of a disease state; particularly to the utilization of massspectrometry to elucidate particular biopolymer markers indicative orpredictive of a particular disease state, and most particularly tospecific biopolymer markers whose up-regulation, down-regulation, orrelative presence in disease vs. normal states has been determined to beuseful in disease state assessment and therapeutic target recognition,development and validation.

BACKGROUND OF THE INVENTION

[0002] Methods utilizing mass spectrometry for the analysis of a targetpolypeptide have been taught wherein the polypeptide is firstsolubilized in an appropriate solution or reagent system. The type ofsolution or reagent system, e.g., comprising an organic or inorganicsolvent, will depend on the properties of the polypeptide and the typeof mass spectrometry performed and are well-known in the art (see, e.g.,Vorm et al. (1994) Anal. Chem. 66:3281 (for MALDI) and Valaskovic et al.(1995) Anal. Chem. 67:3802 (for ESI). Mass spectrometry of peptides isfurther disclosed, e.g., in WO 93/24834 by Chait et al.

[0003] In one prior art embodiment, the solvent is chosen so that therisk that the molecules may be decomposed by the energy introduced forthe vaporization process is considerably reduced, or even fullyexcluded. This can be achieved by embedding the sample in a matrix,which can be an organic compound, e.g., sugar, in particular pentose orhexose, but also polysaccharides such as cellulose. These compounds aredecomposed thermolytically into CO₂ and H₂O so that no residues areformed which might lead to chemical reactions. The matrix can also be aninorganic compound, e.g., nitrate of ammonium which is decomposedpractically without leaving any residues. Use of these and othersolvents are further disclosed in U.S. Pat. No. 5,062,935 by Schlag etal.

[0004] Prior art mass spectrometer formats for use in analyzing thetranslation products include ionization (I) techniques, including butnot limited to matrix assisted laser desorption (MALDI), continuous orpulsed electrospray (ESI) and related methods (e.g., IONSPRAY orTHERMOSPRAY), or massive cluster impact (MCI); these ion sources can bematched with detection formats including linear or non-linear reflectiontime-off-light (TOF), single or multiple quadropole, single or magneticsector, Fourier Transform ion cyclotron resonance (FTICR), ion trap, andcombinations thereof (e.g., ion-trap/time-of-flight). For ionization,numerous matrix/wavelength combinations (MALDI) or solvent combinations(ESI) can be employed. Subattomole levels of protein have been detected,for example, using ESI (Valaskovic, G. A. et al., (1996) Science273:1199-1202) or MALDI (Li, L. et al., (1996) J. Am. Chem. Soc.118:1662-1663) mass spectrometry.

[0005] ES mass spectrometry has been introduced by Fenn et al. (J. Phys.Chem. 88, 4451-59 (1984); PCT Application No. WO 90/14148) and currentapplications are summarized in recent review articles (R. D. Smith etal., Anal. Chem. 62, 882-89 (1990) and B. Ardrey, Electrospray MassSpectrometry, Spectroscopy Europe, 4, 10-18 (1992)). MALDI-TOF massspectrometry has been introduced by Hillenkamp et al. (“Matrix AssistedUV-Laser Desorption/Ionization: A New Approach to Mass Spectrometry ofLarge Biomolecules,” Biological Mass Spectrometry (Burlingame andMcCloskey, editors), Elsevier Science Publishers, Amsterdam, pp. 49-60,1990). With ESI, the determination of molecular weights in femtomoleamounts of sample is very accurate due to the presence of multiple ionpeaks which all could be used for the mass calculation.

[0006] The mass of the target polypeptide determined by massspectrometry is then compared to the mass of a reference polypeptide ofknown identity. In one embodiment, the target polypeptide is apolypeptide containing a number of repeated amino acids directlycorrelated to the number of trinucleotide repeats transcribed/translatedfrom DNA; from its mass alone the number of repeated trinucleotiderepeats in the original DNA which coded it, may be deduced.

[0007] U.S. Pat. No. 6,020,208 utilizes a general category of probeelements (i.e., sample presenting means) with Surfaces Enhanced forLaser Desorption/Ionization (SELDI), within which there are three (3)separate subcategories. The SELDI process is directed toward a samplepresenting means (i.e., probe element surface) with surface-associated(or surface-bound) molecules to promote the attachment (tethering oranchoring) and subsequent detachment of tethered analyte molecules in alight-dependent manner, wherein the said surface molecule(s) areselected from the group consisting of photoactive (photolabile)molecules that participate in the binding (docking, tethering, orcrosslinking) of the analyte molecules to the sample presenting means(by covalent attachment mechanisms or otherwise).

[0008] PCT/EP/04396 teaches a process for determining the status of anorganism by peptide measurement. The reference teaches the measurementof peptides in a sample of the organism which contains both high and lowmolecular weight peptides and acts as an indicator of the organism'sstatus. The reference concentrates on the measurement of low molecularweight peptides, i.e. below 30,000 Daltons, whose distribution serves asa representative cross-section of defined controls. Contrary to themethodology of the instant invention, the '396 patent strives todetermine the status of a healthy organism, i.e. a “normal” and then usethis as a reference to differentiate disease states. The presentinventors do not attempt to develop a reference “normal”, but ratherstrive to specify particular markers whose presence, absence or relativestrength/concentration in disease vs. normal is diagnostic of at leastone specific disease state or whose up-regulation or down-regulation ispredictive of at least one specific disease state, whereby the presenceof said marker serves as a positive indicator useful in distinguishingdisease state. This leads to a simple method of analysis which caneasily be performed by an untrained individual, since there is apositive correlation of data. On the contrary, the '396 patent requiresa complicated analysis by a highly trained individual to determinedisease state versus the perception of non-disease or normal physiology.

[0009] Richter et al, Journal of Chromatography B, 726(1999) 25-35,refer to a database established from human hemofiltrate comprised of amass database and a sequence database. The goal of Richter et al was toanalyze the composition of the peptide fraction in human blood. UsingMALDI-TOF, over 20,000 molecular masses were detected representing anestimated 5,000 different peptides. The conclusion of the study was thatthe hemofiltrate (HF) represented the peptide composition of plasma. Nocorrelation of peptides with relation to normal and/or disease states ismade.

[0010] As used herein, “analyte” refers to any atom and/or molecule;including their complexes and fragment ions. The term may refer to asingle component or a set of components. In the case of biologicalmolecules/macromolecules or “biopolymers”, such analytes include but arenot limited to: polypeptides, polynucleotides, proteins, peptides,antibodies, DNA, RNA, carbohydrates, steroids, and lipids, and anydetectable moiety thereof, e.g. immunologically detectable fragments.Note that most important biomolecules under investigation for theirinvolvement in the structure or regulation of life processes are quitelarge (typically several thousand times larger than H₂O).

[0011] As used herein, the term “molecular ions” refers to molecules inthe charged or ionized state, typically by the addition or loss of oneor more protons (H⁺).

[0012] As used herein, the term “molecular fragmentation” or “fragmentions” refers to breakdown products of analyte molecules caused, forexample, during laser-induced desorption (especially in the absence ofadded matrix).

[0013] As used herein, the term “solid phase” refers to the condition ofbeing in the solid state, for example, on the probe element surface.

[0014] As used herein, “gas” or “vapor phase” refers to molecules in thegaseous state (i.e., in vacuo for mass spectrometry).

[0015] As used herein, the term “analyte desorption/ionization” refersto the transition of analytes from the solid phase to the gas phase asions. Note that the successful desorption/ionization of large, intactmolecular ions by laser desorption is relatively recent (circa 1988)—thebig breakthrough was the chance discovery of an appropriate matrix(nicotinic acid).

[0016] As used herein, the term “gas phase molecular ions” refers tothose ions that enter into the gas phase. Note that large molecular massions such as proteins (typical mass=60,000 to 70,000 times the mass of asingle proton) are typically not volatile (i.e., they do not normallyenter into the gas or vapor phase). However, in the procedure of thepresent invention, large molecular mass ions such as proteins do enterthe gas or vapor phase.

[0017] As used herein in the case of MALDI, the term “matrix” refers toany one of several small, acidic, light absorbing chemicals (e.g., CHCA(alpha-cyano-4-hydroxy-cinnamic acid), nicotinic or sinapinic acid) thatis mixed in solution with the analyte in such a manner so that, upondrying on the probe element, the crystalline matrix-embedded analytemolecules are successfully desorbed (by laser irradiation) and ionizedfrom the solid phase (crystals) into the gaseous or vapor phase andaccelerated as intact molecular ions. For the MALDI process to besuccessful, analyte is mixed with a freshly prepared solution of thechemical matrix (e.g., 10,000:1 matrix:analyte) and placed on the inertprobe element surface to air dry just before the mass spectrometricanalysis. The large fold molar excess of matrix, present atconcentrations near saturation, facilitates crystal formation andentrapment of analyte.

[0018] As used herein, “energy absorbing molecules (EAM)” refers to anyone of several small, light absorbing chemicals that, when presented onthe surface of a probe, facilitate the neat desorption of molecules fromthe solid phase (i.e., surface) into the gaseous or vapor phase forsubsequent acceleration as intact molecular ions. The term EAM ispreferred, especially in reference to SELDI. Note that analytedesorption by the SELDI process is defined as a surface-dependentprocess (i.e., neat analyte may be placed on a surface composed of boundEAM or EAM and analyte may be mixed prior to placement on a surface). Incontrast, MALDI is presently thought to facilitate analyte desorption bya volcanic eruption-type process that “throws” the entire surface intothe gas phase. Furthermore, note that some EAM when used as freechemicals to embed analyte molecules as described for the MALDI processwill not work (i.e., they do not promote molecular desorption, thus theyare not suitable matrix molecules).

[0019] As used herein, “probe element” or “sample presenting device”refers to an element having the following properties: it is inert (forexample, typically stainless steel) and active (probe elements withsurfaces enhanced to contain EAM and/or molecular capture devices).

[0020] As used herein, “MALDI” refers to Matrix-Assisted LaserDesorption/Ionization.

[0021] As used herein, “TOF” stands for Time-of-Flight.

[0022] As used herein, “MS” refers to Mass Spectrometry.

[0023] As used herein, “MS/MS” refers to multiple sequential massspectrometry.

[0024] As used herein “MALDI-TOF MS” refers to Matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry.

[0025] As used herein, “ESI” is an abbreviation for electrosprayionization.

[0026] As used herein, “chemical bonds” is used simply as an attempt todistinguish a rational, deliberate, and knowledgeable manipulation ofknown classes of chemical interactions from the poorly defined kind ofgeneral adherence observed when one chemical substance (e.g., matrix) isplaced on another substance (e.g., an inert probe element surface).Types of defined chemical bonds include electrostatic or ionic (+/−)bonds (e.g., between a positively and negatively charged groups on aprotein surface), covalent bonds (very strong or “permanent” bondsresulting from true electron sharing), coordinate covalent bonds (e.g.,between electron donor groups in proteins and transition metal ions suchas copper or iron), and hydrophobic interactions (such as between twononcharged groups), weak dipole and London force or induced dipoleinteractions.

[0027] As used herein, “electron donor groups” refers to the case ofbiochemistry, where atoms in biomolecules (e.g, N, S, O) “donate” orshare electrons with electron poor groups (e.g., Cu ions and othertransition metal ions).

[0028] As used herein, the term “biopolymer markers indicative orpredictive of a disease state” is interpreted to mean that a biopolymermarker which is strongly present in a normal individual, but isdown-regulated in disease is predictive of said disease; whilealternatively, a biopolymer marker which is strongly present in adisease state, but is down-regulated in normal individuals, isindicative of said disease state. Biopolymer markers which are presentin both disease and normal states are indicative/predictive based upontheir relative strengths in disease vs. normal, along with theobservation regarding when their signal strengthens/weakens relative todisease manifestation or progression.

[0029] As used herein, the term “disease state assessment” isinterpreted to mean quantitative or qualitative determination of thepresence/absence of the disease, with or without an ability to determineseverity, rapidity of onset, or resolution of the disease state, e.g. areturn to a normal physiological state.

[0030] As used herein, the term “therapeutic target recognition,development, and validation” refers to any concept or method whichenables an artisan to recognize, develop, or validate the efficacy of atherapeutic moiety which is effected in conjunction with a chemical orphysical interaction with one or more of the biopolymer markers of theinstant invention.

[0031] As used herein, the term “polypeptide” is interpreted to mean apolymer composed of amino acid residues, related naturally occurringstructural variants, and synthetic non-naturally occurring analogsthereof linked via peptide bonds, related naturally occurring structuralvariants, and synthetic non-naturally occurring analogs thereof.Synthetic polypeptides can be synthesized, for example, using anautomated polypeptide synthesizer. The term “protein” typically refersto large polypeptides. The term “peptide” typically refers to shortpolypeptides. “Polypeptide(s)” refers to any peptide or proteincomprising two or more amino acids joined to each other by peptide bondsor modified peptide bonds. “Polypeptide(s)” refers to both short chains,commonly referred to as peptides, oligopeptides and oligomers and tolonger chains generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene encoded amino acids.“Polypeptide(s)” include those modified either by natural processes,such as processing and other post-translational modifications, but alsoby chemical modification techniques. Such modifications are welldescribed in basic texts and in more detailed monographs, as well as ina voluminous research literature, and they are well-known to those ofskill in the art. It will be appreciated that the same type ofmodification may be present in the same or varying degree at severalsites in a given polypeptide. Also, a given polypeptide may contain manytypes of modifications. Modifications can occur anywhere in apolypeptide, including the peptide backbone, the amino acid side-chains,and the amino or carboxyl termini. Modifications include, for example,acetylation, acylation, ADPribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-link formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, selenoylation, sulfation, transfer-RNA mediatedaddition of amino acids to proteins, such as arginylation, andubiquitination. See, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993) and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched orcyclic, with or without branching. Cyclic, branched and branchedcircular polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

[0032] As used herein, the term “polynucleotide” is interpreted to meana polymer composed of nucleotide units. Polynucleotides includenaturally occurring nucleic acids, such as deoxyribonucleic acid (“DNA”)and ribonucleic acid (“RNA”) as well as nucleic acid analogs. Nucleicacid analogs include those which include non-naturally occurring bases,nucleotides that engage in linkages with other nucleotides other thanthe naturally occurring phosphodiester bond or which include basesattached through linkages other than phosphodiester bonds. Thus,nucleotide analogs include, for example and without limitation,phosphorothioates, phosphorodithioates, phosphorotriesters,phosphoramidates, boranophosphates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs),and the like. Such polynucleotides can be synthesized, for example,using an automated DNA synthesizer. The term “nucleic acid” typicallyrefers to large polynucleotides. The term “oligonucleotide” typicallyrefers to short polynucleotides, generally no greater than about 50nucleotides. It will be understood that when a nucleotide sequence isrepresented by a DNA sequence (i.e., A, T, G, C), this also includes anRNA sequence (i.e., A, U, G, C) in which “U” replaces T.

[0033] As used herein, the term “detectable moiety” or a “label” refersto a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³²P, ³⁵S, fluorescent dyes, electron-dense reagents,enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin,dioxigenin, haptens and proteins for which antisera or monoclonalantibodies are available, or nucleic acid molecules with a sequencecomplementary to a target. The detectable moiety often generates ameasurable signal, such as a radioactive, chromogenic, or fluorescentsignal, that can be used to quantitate the amount of bound detectablemoiety in a sample. The detectable moiety can be incorporated in orattached to a primer or probe either covalently, or through ionic, vander Waals or hydrogen bonds, e.g., incorporation of radioactivenucleotides, or biotinylated nucleotides that are recognized bystreptavadin. The detectable moiety may be directly or indirectlydetectable. Indirect detection can involve the binding of a seconddirectly or indirectly detectable moiety to the detectable moiety. Forexample, the detectable moiety can be the ligand of a binding partner,such as biotin, which is a binding partner for streptavadin, or anucleotide sequence, which is the binding partner for a complementarysequence, to which it can specifically hybridize. The binding partnermay itself be directly detectable, for example, an antibody may beitself labeled with a fluorescent molecule. The binding partner also maybe indirectly detectable, for example, a nucleic acid having acomplementary nucleotide sequence can be a part of a branched DNAmolecule that is in turn detectable through hybridization with otherlabeled nucleic acid molecules. (See, e.g., P. D. Fahrlander and A.Klausner, Bio/Technology (1988) 6:1165.) Quantitation of the signal isachieved by, e.g., scintillation counting, densitometry, or flowcytometry.

[0034] As used herein, the term “antibody or antibodies” includespolyclonal and monoclonal antibodies of any isotype (IgA, IgG, IgE, IgD,IgM), or an antigen-binding portion thereof, including but not limitedto F(ab) and Fv fragments, single chain antibodies, chimeric antibodies,humanized antibodies, and a Fab expression library. “Antibody” refers toa polypeptide ligand substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, which specifically binds andrecognizes an epitope (e.g., an antigen). The recognizedimmunoglobulin—genes include the kappa and lambda light chain constantregion genes, the alpha, gamma, delta, epsilon and mu heavy chainconstant region genes, and the myriad immunoglobulin variable regiongenes. Antibodies exist, e.g., as intact immunoglobulins or as a numberof well characterized fragments produced by digestion with variouspeptidases. This includes, e.g., Fab′ and F(ab)′₂ fragments. The term“antibody,” as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies. It also includes polyclonalantibodies, monoclonal antibodies, chimeric antibodies and humanizedantibodies. “Fc” portion of an antibody refers to that portion of animmunoglobulin heavy chain that comprises one or more heavy chainconstant region domains, CH, CH₂ and CH₃, but does not include the heavychain variable region.

[0035] As used herein, the term “moieties” refers to an indefiniteportion of a sample.

[0036] A “ligand” is a compound that specifically binds to a targetmolecule.

[0037] A “receptor” is a compound or portion of a structure thatspecifically binds to a ligand.

[0038] A ligand or a receptor (e.g., an antibody) “specifically bindsto” or “is specifically immunoreactive with” a compound analyte when theligand or receptor functions in a binding reaction which isdeterminative of the presence of the analyte in a sample ofheterogeneous compounds. Thus, under designated assay (e.g.,immunoassay) conditions, the ligand or receptor binds preferentially toa particular analyte and does not bind in a significant amount to othercompounds present in the sample. For example, a polynucleotidespecifically binds under hybridization conditions to an analytepolynucleotide comprising a complementary sequence; an antibodyspecifically binds under immunoassay conditions to an antigen analytebearing an epitope against which the antibody was raised; and anadsorbent specifically binds to an analyte under proper elutionconditions.

[0039] As used herein, the term “pharmaceutically effective carrier”refers to any solid or liquid material which may be used in creatingformulations that further include active ingredients of the instantinvention, e.g. biopolymer markers or therapeutics, for administrationto a patient.

[0040] As used herein, the term “agent” is interpreted to mean achemical compound, a mixture of chemical compounds, a sample ofundetermined composition, a combinatorial small molecule array, abiological macromolecule, a bacteriophage peptide display library, abacteriophage antibody (e.g., scFv) display library, a polysome peptidedisplay library, or an extract made from biological materials such asbacteria, plants, fungi, or animal cells or tissues. Suitable techniquesinvolve selection of libraries of recombinant antibodies in phage orsimilar vectors. See, Huse et al. (1989) Science 246: 1275-1281; andWard et al. (1989) Nature 341: 544-546. The protocol described by Huseis rendered more efficient in combination with phage display technology.See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047.

[0041] As used herein, the term “isolated” is interpreted to meanaltered “by the hand of man” from its natural state, i.e., if it occursin nature, it has been changed or removed from its original environment,or both. For example, a polynucleotide or a polypeptide naturallypresent in a living organism is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated”, as the term is employed herein.

[0042] As used herein, the term “variant” is interpreted to mean apolynucleotide or polypeptide that differs from a referencepolynucleotide or polypeptide respectively, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques, by direct synthesis,and by other recombinant methods known to skilled artisans.

[0043] As used herein, the term “biopolymer marker” refers to a polymerof biological origin, e.g. polypeptides, polynucleotides,polysaccharides or polyglycerides (e.g., dior tri-glycerides), and mayinclude any fragment, e.g. immunologically reactive fragments, variantsor moieties thereof.

[0044] As used herein, the term “fragment” refers to the products of thechemical, enzymatic, or physical breakdown of an analyte. Fragments maybe in a neutral or ionic state.

[0045] As used herein, the term “therapeutic avenues” is interpreted tomean any agents, modalities, synthesized compounds, etc., which interactwith a biopolymer marker in any manner that facilitates a therapeuticbenefit, including immunotherapeutic intervention, e.g. modalities suchas administration of an immunologically reactive moiety capable ofaltering the course, progression and/or manifestation of the disease, asa result of interfering with the disease manifestation process, forexample, at the early stages focused upon by the identification of thedisease, such as by supplying a moiety capable of modifying thepathogenicity of lymphocytes specific for the biopolymer marker orrelated components.

[0046] As used herein, the term “interacting with a biopolymer marker”includes any process by which a biopolymer marker may physically orchemically relate with an organism, particularly when this interactionresults in the development of therapeutic avenues or in modulation ofthe disease state.

[0047] As used herein, the term “therapeutic targets” may thus bedefined as those analytes which are capable of exerting a modulatingforce, wherein “modulation” is defined as an alteration in functioninclusive of activity, synthesis, production, and circulating levels.Thus, modulation effects the level or physiological activity of at leastone particular disease related biopolymer marker or any compound orbiomolecule whose presence, level or activity is linked either directlyor indirectly, to an alteration of the presence, level, activity orgeneric function of the biopolymer marker, and may includepharmaceutical agents, biomolecules that bind to the biopolymer markers,or biomolecules or complexes to which the biopolymer markers bind. Thebinding of the biopolymer markers and the therapeutic moiety may resultin activation (agonist), inhibition (antagonist), or an increase ordecrease in activity or production (modulator) of the biopolymer markersor the bound moiety. Examples of such therapeutic moieties include, butare not limited to, antibodies, oligonucleotides, proteins (e.g.,receptors), RNA, DNA, enzymes, peptides or small molecules. With regardto immunotherapeutic moieties, such a moiety may be defined as aneffective analog for a major epitope peptide which has the ability toreduce the pathogenicity of key lymphocytes which are specific for thenative epitope. An analog is defined as having structural similarity butnot identity in peptide sequencing able to be recognized by T-cellsspontaneously arising and targeting the endogeneous self epitope. Acritical function of this analog is an altered T-cell activation whichleads to T-cell anergy or death.

[0048] With the advent of mass spectrometric methods such as MALDI andSELDI and ESI, researchers have begun to utilize a tool that holds thepromise of uncovering countless biopolymers which result fromtranslation, transcription and post-translational transcription ofproteins from the entire genome.

[0049] Operating upon the principles of retentate chromatography, SELDIMS involves the adsorption of proteins, based upon theirphysico-chemical properties at a given pH and salt concentration,followed by selectively desorbing proteins from the surface by varyingpH, salt, or organic solvent concentration. After selective desorption,the proteins retained on the SELDI surface, the “chip”, can be analyzedusing the CIPHERGEN protein detection system, or an equivalent thereof.Retentate chromatography is limited, however, by the fact that ifunfractionated body fluids, e.g. blood, blood products, urine, saliva,cerebrospinal fluid, luymph and the like, along with tissue samples, areapplied to the adsorbent surfaces, the biopolymers present in thegreatest abundance will compete for all the available binding sites andthereby prevent or preclude less abundant biopolymers from interactingwith them, thereby reducing or eliminating the diversity of biopolymerswhich are readily ascertainable.

[0050] If a process could be devised for maximizing the diversity ofbiopolymers discernable from a sample, the ability of researchers toaccurately determine the relevance of such biopolymers with relation toone or more disease states would be immeasurably enhanced.

SUMMARY OF THE INVENTION

[0051] The instant invention is characterized by the use of acombination of preparatory steps, e.g. chromatography and 1-D tricinepolyacrylamide gel electrophoresis. Subsequent to which the gel isstained, e.g. with Coomasie blue, silver or rubidium. Next, bands areselected from the gels for further study. Tryptic digestion of each bandfollows, concluding with the extraction of tryptic peptides from thedigest. This extraction may be accomplished utilizing C18 ZIPTIPs, ororganic extract and dry technique followed by MALDI Qq TOF (MaldiQuadrupole Quadrupole Time of Flight) processing.

[0052] Additional methodologies may include SELDI MS, 2-D geltechnology, MALDI MS/MS and time-of-flight detection procedures tomaximize the diversity of biopolymers which are verifiable within aparticular sample. The cohort of biopolymers verified within a sample isthen compared to develop data indicating their presence, absence orrelative strength/concentration in disease vs normal controls, andfurther studied to determine whether the up-regulation ordown-regulation of a single biopolymer or group of biopolymers isindicative of a disease state or predictive of the development of saiddisease state. Additionally, biopolymers recognized as being indicativeor predictive of a disease state in accordance with the instantinvention are useful in therapeutic intervention, e.g. as therapeuticmodalities in their own right, in the course of therapeutic targetrecognition, in the development and validation of efficacioustherapeutic modalities, e.g when interrogating or developing phagedisplay libraries, and as ligands or receptors for use in conjunctionwith therapeutic intervention.

[0053] Although all manner of biomarkers related to all diseaseconditions are deemed to be within the purview of the instant inventionand methodology, particular significance was given to those markers anddiseases associated with the complement system, cognitive diseases, e.g.Alzheimer's disease and Syndrome X and diseases related thereto.

[0054] The complement system is an important part of non-clonal orinnate immunity that collaborates with acquired immunity to destroyinvading pathogens and to facilitate the clearance of immune complexesfrom the system. This system is the major effector of the humoral branchof the immune system, consisting of nearly 30 serum and membraneproteins. The proteins and glycoproteins composing the complement systemare synthesized largely by liver hepatocytes. Activation of thecomplement system involves a sequential enzyme cascade in which theproenzyme product of one step becomes the enzyme catalyst of the nextstep. Complement activation can occur via two pathways: the classicaland the alternative. The classical pathway is commonly initiated by theformation of soluble antigen-antibody complexes or by the binding ofantibody to antigen on a suitable target, such as a bacterial cell. Thealternative pathway is generally initiated by various cell-surfaceconstituents that are foreign to the host. Each complement component isdesignated by numerals (C1-C9), by letter symbols, or by trivial names.After a component is activated, the peptide fragments are denoted bysmall letters. The complement fragments interact with one another toform functional complexes. Ultimately, foreign cells are destroyedthrough the process of a membrane-attack complex mediated lysis.

[0055] The C4 component of the complement system is involved in theclassical activation pathway. It is a glycoprotein containing threepolypeptide chains (α, β, and γ). C4 is a substrate of component C1s andis activated when C1s hydrolyzes a small fragment (C4a) from the aminoterminus of the α chain, exposing a binding site on the larger fragment(C4b).

[0056] The native C3 component consists of two polypeptide chains, α andβ. As a serum protein, C3 is involved in the alternative pathway. SerumC3, which contains an unstable thioester bond, is subject to slowspontaneous hydrolysis into C3a and C3b. The C3f component is involvedin the regulation required of the complement system which confines thereaction to designated targets. During the regulation process, C3b iscleaved into two parts: C3bi and C3f. C3bi is a membrane-boundintermediate wherein C3f is a free diffusible (soluble) component.

[0057] Complement components have been implicated in the pathogenesis ofseveral disease conditions. C3 deficiencies have the most severeclinical manifestations, such as recurrent bacterial infections andimmune-complex diseases, reflecting the central role of C3. The rapidprofusion of C3f moieties and resultant “accidental” lysis of normalcells mediated thereby gives rise to a host of auto-immune reactions.The ability to understand and control these mechanisms, along with theirattendant consequences, will enable practitioners to develop bothdiagnostic and therapeutic avenues by which to thwart these maladies.

[0058] In the course of defining a plurality of disease specific markersequences, special significance was given to markers which wereevidentiary of a particular disease state or with conditions associatedwith Syndrome-X. Syndrome-X is a multifaceted syndrome, which occursfrequently in the general population. A large segment of the adultpopulation of industrialized countries develops this metabolic syndrome,produced by genetic, hormonal and lifestyle factors such as obesity,physical inactivity and certain nutrient excesses. This disease ischaracterized by the clustering of insulin resistance andhyperinsulinemia, and is often associated with dyslipidemia (atherogenicplasma lipid profile), essential hypertension, abdominal (visceral)obesity, glucose intolerance or noninsulin-dependent diabetes mellitusand an increased risk of cardiovascular events. Abnormalities of bloodcoagulation (higher plasminogen activator inhibitor type I andfibrinogen levels), hyperuricemia and microalbuminuria have also beenfound in metabolic syndrome-X.

[0059] The instant inventors view the Syndrome X continuum in itscardiovascular light, while acknowledging its important metaboliccomponent. The first stage of Syndrome X consists of insulin resistance,abnormal blood lipids (cholesterol, triglycerides and free fatty acids),obesity, and high blood pressure (hypertension). Any one of these fourfirst stage conditions signals the start of Syndrome X.

[0060] Each first stage Syndrome X condition risks leading to another.For example, increased insulin production is associated with high bloodfat levels, high blood pressure, and obesity. Furthermore, the effectsof the first stage conditions are additive; an increase in the number ofconditions causes an increase in the risk of developing more seriousdiseases on the Syndrome X continuum.

[0061] A patient who begins the Syndrome X continuum risks spiralinginto a maze of increasingly deadly diseases. The next stages of theSyndrome X continuum lead to overt diabetes, kidney failure, and heartfailure, with the possibility of stroke and heart attack at any time.Syndrome X is a dangerous continuum, and preventative medicine is thebest defense. Diseases are currently most easily diagnosed in theirlater stages, but controlling them at a late stage is extremelydifficult. Disease prevention is much more effective at an earlierstage.

[0062] In a further contemplated embodiment of the invention, samplesmay be taken from a patient at one point in time, as a single sample oras multiple samples, or at different points in time such that analysisis carried out on multiple samples for ongoing analysis. Typically, afirst sample is taken from a patient upon presentation with possiblesymptoms of a disease and analyzed according to the invention.Subsequently, some period of time after presentation, for example, about3-6 months after the first presentation, a second sample is taken andanalyzed according to the invention. The data can be used, by way ofexample, to diagnose or monitor a disease state, determine riskassessment, identify therapeutic avenues, or determine the therapeuticvalue of an agent such as a pharmaceutical.

[0063] Subsequent to the isolation of particular disease state markersequences as taught by the instant invention, the promulgation ofvarious forms of risk assessment tests are contemplated which will allowphysicians to identify asymptomatic patients before they suffer anirreversible event such as diabetes, kidney failure, and heart failure,and enable effective disease management and preventative medicine.Additionally, the specific diagnostic tests which evolve from thismethodology provide a tool for rapidly and accurately diagnosing acuteSyndrome X events such as heart attack and stroke, and facilitatetreatment.

[0064] More particularly, biopolymer markers elucidated viamethodologies of the instant invention find utility related to broadareas of disease therapeutics. Such therapeutic avenues include, but arenot limited to:

[0065] 1) utilization and recognition of said biopolymer markers,variants or moieties thereof as direct therapeutic modalities, eitheralone or in conjunction with an effective amount of a pharmaceuticallyeffective carrier;

[0066] 2) validation of therapeutic modalities or disease preventativeagents as a function of biopolymer marker presence or concentration;

[0067] 3) treatment or prevention of a disease state by formation ofdisease intervention modalities; e.g. formation of biopolymer/ligandconjugates which intervene at receptor sites to prevent, delay orreverse a disease process;

[0068] 4) use of biopolymer markers or moieties thereof as a means ofelucidating therapeutically viable agents, e.g. from a bacteriophagepeptide display library, a bacteriophage antibody library or the like;

[0069] 5) instigation of a therapeutic immunological response; and

[0070] 6) synthesis of molecular structures related to said biopolymermarkers, moieties or variants thereof which are constructed and arrangedto therapeutically intervene in the disease process.

[0071] A process for identifying or developing therapeutic avenuesrelated to a disease state utilizing any of the above examples mayfollow results obtained from conducting an analysis inclusive ofinteracting with a biopolymer including the sequence of the particulardisease specific marker or at least one analyte thereof of the presentinvention. Such treatment or prevention of a disease state by formationof disease intervention modalities may be by the formation ofbiopolymer/ligand conjugates which intervene at receptor sites toprevent, delay, or reverse a disease process. In addition, a means ofelucidating therapeutically viable agents may include the use of abacteriophage peptide display library or a bacteriophage antibodylibrary. The therapeutic avenues may regulate the presence or absence ofthe biopolymer including the sequence of the particular disease specificmarker or at least one analyte thereof in the present invention.

[0072] Accordingly, it is an objective of the instant invention todefine a disease specific biopolymer marker sequence which is useful inevidencing and categorizing at least one particular disease state.

[0073] It is an additional objective of the instant invention to developmethods and means of disease therapy, including but not limited to:

[0074] 1) utilization and recognition of said biopolymer markers,variants or moieties thereof as direct therapeutic modalities, eitheralone or in conjunction with an effective amount of a pharmaceuticallyeffective carrier;

[0075] 2) validation of therapeutic modalities or disease preventativeagents as a function of biopolymer marker presence or concentration;

[0076] 3) treatment or prevention of a disease state by formation ofdisease intervention modalities; e.g. formation of biopolymer/ligandconjugates which intervene at receptor sites to prevent, delay orreverse a disease process;

[0077] 4) use of biopolymer markers or moieties thereof as a means ofelucidating therapeutically viable agents, e.g. from a bacteriophagepeptide display library, a bacteriophage antibody library or the like;

[0078] 5) instigation of a therapeutic immunological response; and

[0079] 6) synthesis of molecular structures related to said biopolymermarkers, moieties or variants thereof which are constructed and arrangedto therapeutically intervene in the disease process, e.g. by directlydetermining the three-dimensional structure of said biopolymer markerdirectly from an amino acid sequence thereof.

[0080] It is another objective of the instant invention to evaluatesamples containing a plurality of biopolymers for the presence ofdisease specific biopolymer marker sequences (disease specific markers)which evidence a link to at least one specific disease state.

[0081] It is a further objective of the instant invention to elucidateessentially all biopolymeric markers, moieties or variants thereofcontained within said samples, whereby particularly significant moietiesmay be identified.

[0082] It is a further objective of the instant invention provide atleast one purified antibody which is specific to said disease specificmarker sequence.

[0083] It is yet another objective of the instant invention to teach amonoclonal antibody which is specific to said disease specific markersequence.

[0084] It is a still further objective of the invention to teachpolyclonal antibodies raised against said disease specific marker.

[0085] It is yet an additional objective of the instant invention toteach a diagnostic kit for determining the presence, concentration, orrelative strength/concentration of said disease specific marker.

[0086] It is a still further objective of the instant invention to teachmethods for characterizing disease state based upon the identificationof said disease specific marker.

[0087] Other objects and advantages of this invention will becomeapparent from the following description taken in conjunction with theaccompanying drawings wherein are set forth, by way of illustration andexample, certain embodiments of this invention. The drawings constitutea part of this specification and include exemplary embodiments of thepresent invention and illustrate various objects and features thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0088]FIG. 1 is a photograph of a tricine gel DEAE 2 (Elution) comparingInsulin Resistance versus Normal;

[0089]FIG. 2 is a trypsin digested spectra graph depicting the ion 1301;

[0090]FIG. 3 is a trypsin digested spectra graph depicting the ion 1187.

DETAILED DESCRIPTION OF THE INVENTION

[0091] In earlier work, for example in U.S. patent application Ser. No.09/846330 filed Apr. 30, 2000, the contents of which is hereinincorporated by reference, raw sera was obtained and mixed with formicacid and extracted the peptides with C18 reversed phase ZIPTIPs.

[0092] In the instantly disclosed invention, we deal with proteinsgenerally having a molecular weight of about 20 kD or more. In general,proteins of greater than 20 kD can reliably be fragmented by trypsin orother enzymes. The instant technology incorporates sufficientsensitivity to deal with even the low production of peptides fromproteins less than 20 kD clipped from gel.

[0093] Proteins differ from peptides in that they cannot be effectivelyresolved by time of flight MS and they are too large (>3 kD) to beeffectively fragmented by collision with gases. The most commonly usedsolution to these problems is to resolve the proteins by polyacrylamidegel electrophoresis followed by staining with silver, or coomasiebrilliant blue or rubidium dyes or counter staining with Zinc-SDScomplexes. Once the proteins have been resolved and visualized withstains the proteins that differ between disease states can then beexcised from the gel and the protein purified in the 1-D gel band or 2-Dgel spot can be cleaved into fragments less that 3 kD by proteolyticenzymes. Once protein has been resolved by gel and cleaved by enzymes,the protein is considered in the form of peptides and therefore can bedealt with as per earlier work (09/846330). The peptide is eithercollected and purified with C18 reversed phase chromatography or by someother form of chromatography prior to reversed phase separation. Thepeptide can also be collected in ammonium carbonate buffer that issubsequently evolved by reaction with acid or by removal in organicsolvents.

[0094] Once the peptides are collected they can be sequenced, e.g. witha MALDI-Qq-TOF but also with a TOF-TOF, and ESI-Q-TOF or an ION-TRAP.Other types of MS analysis which may be employed are SELDI MS and MS/MS.The peptides are fragments of the original protein. The peptides aresequenced by fragmentation to produced a spectrum composed of the partsof the peptide. The peptide fragments can be produced by a strongionization energy with a laser, temperature, electron capture, collisionbetween the peptides themselves or with other objects such as gasmolecules. The spacing in terms of mass between the parts of thepeptides is a fragmentation pattern. The fragmentation pattern of eachpeptide from the starting mass to the last remaining amino acid (fromeither end) is unique.

[0095] The human genome contains the genes that encode all proteins. Theproteolytic cut sites within all these proteins can be predicted fromthe translated amino acid sequence. The mass of the peptides that resultfrom the predicting cut sites can be calculated. Similarly, thefragmentation pattern from each hypothetical peptide can be predicted.Thus, we can conceptually digest the proteins within the human proteomeand fragment them.

[0096] When a peptide has been “sequenced” it is understood that thepeptide fragment has been purified by one of the methods above, i.e.Time of flight (TOF) or by chromatography, before fragmenting it withgas to produce the peptide fragments. The original peptide mass andfragmentation pattern obtained is then fit to those from the theoreticaldigestion and fragmentation of the genome. The peptide that best matchesthe theoretical peptides and fragments and is biologically possible,i.e. a potential human blood-borne protein, is thus identified. It ispossible to identify plural targets in this fashion.

[0097] Following are exemplary, but non-limiting examples of preparatoryprotocols useful in the process of the instant invention.

[0098] Preparatory Protocols:

[0099] Any of these protocols may be selected from a column flow-throughstream, a column elution stream, or a column scrub stream.

[0100] Hi Q is a strong anion exchanger made of methyl acrylateco-polymer with the functional group: —N⁺(CH₃)₂;

[0101] Hi S is a strong cation exchanger made of methyl acrylateco-polymer with the functional group: —SO₃ ⁻;

[0102] DEAE is diethylaminoethyl which is a weak cation exchanger madeof methyl acrylate co-polymer with the functional group —N⁺(C₂H₅)₂;

[0103] PS is phenyl sepharose;

[0104] BS is butyl sepharose.

[0105] Note that the supports, i.e. methyl acrylate and sepharose aredifferent, but non-limiting examples, as the same functional group ondifferent supports will function, albeit possibly with differenteffects.

[0106] DEAE Column Protocol:

[0107] 1)Cast 200 μl of 50% slurry;

[0108] 2)Equilibrate column in 5 bed volumes of 50 mM tricine pH 8.8(binding buffer);

[0109] 3)Dissolve 25 μl of sera in 475 μl of binding buffer;

[0110] 4)Wash column in 5 bed volumes of binding buffer;

[0111] 5)Elute column in 120 μl of 0.4 M Phosphate buffer (PB) pH 6.1;

[0112] 6)Elute column in 120 μl of 50 mM citrate buffer pH 4.2;

[0113] 7)Scrub column with 120 μl sequentially with each of 0.1% triton,1.0% triton and 2% SDS in 62.5 mM Tris pH 6.8.

[0114] Butyl Sepharose Column Protocol:

[0115] 1)Cast 150 μl bed volume column;

[0116] 2)Equilibrate column in 5 bed volumes of 1.7 M (NH₄)₂SO₄ in 50 mMPB pH 7.0 (binding buffer);

[0117] 3)Dissolve 35 μl of sera in 465 μl of binding buffer and apply;

[0118] 4)Wash column in 5 bed volumes of binding buffer;

[0119] 5)Elute column in 120 μl of 0.4 M (NH₄)₂SO₄ in 50 mM PB pH 7.0;

[0120] 6)Elute column in 120 μl of 50 mM PB pH 7.0;

[0121] 7)Scrub column with 120 μl sequentially with each of 0.1% triton,1.0% triton and 2% SDS in 62.5 mM Tris pH 6.8.

[0122] Phenyl Sepharose Column Protocol:

[0123] 1)Cast 150 μl bed volume column;

[0124] 2)Equilibrate column in 5 bed volumes of 1.7 M (NH₄)₂SO₄ in 50 mMPB pH 7.0 (binding buffer);

[0125] 3)Dissolve 35 μl of sera in 465 μl of binding buffer and apply;

[0126] 4)Wash column in 5 bed volumes of binding buffer;

[0127] 5)Elute column in 120 μl of 0.2 M (NH₄)₂SO₄ in 50 mM PB pH 7.0;

[0128] 6)Elute column in 120 μl of 50 mM PB pH 7.0;

[0129] 7)Scrub column with 120 μl sequentially with each of 0.1% triton,1.0% triton and 2% SDS in 62.5 mM Tris pH 6.8.

[0130] HiO Anion Exchange Mini Column Protocol:

[0131] 1)Dilute sera in sample/running buffer;

[0132] 2)Add HiQ resin to column and remove any air bubbles;

[0133] 3)Add ultrafiltered (UF) water to aid in column packing;

[0134] 4)Add sample/running buffer to equilibrate column;

[0135] 5)Add diluted sera;

[0136] 6)Collect all the flow-through fraction in Eppendorf tubes untillevel is at resin;

[0137] 7)Add sample/running buffer to wash column;

[0138] 8)Add elution buffer and collect elution in Eppendorf tubes.

[0139] HiS Cation Exchange Mini Column Protocol:

[0140] 1)Dilute sera in sample/running buffer;

[0141] 2)Add HiS resin to column and remove any air bubbles;

[0142] 3)Add UF water to aid in column packing;

[0143] 4)Add sample/running buffer to equilibrate column for sampleloading;

[0144] 5)Add diluted sera to column;

[0145] 6)Collect all flow through fractions in Eppendorf tubes untillevel is at resin;

[0146] 7)Add sample/running buffer to wash column;

[0147] 8)Add elution buffer and collect elution in Eppendorf tubes.

[0148] Illustrative of the various buffering compositions useful in thistechnique are:

[0149] Sample/Running buffers: including but not limited to Bicinebuffers of various molarities, pH's, NaCl content, Bis-Tris buffers ofvarious molarities, pH's, NaCl content, Diethanolamine of variousmolarities, pH's, NaCl content, Diethylamine of various molarities,pH's, NaCl content, Imidazole of various molarities, pH's, NaCl content,Tricine of various molarities, pH's, NaCl content, Triethanolamine ofvarious molarities, pH's, NaCl content, Tris of various molarities,pH's, NaCl content. Elution Buffer: Acetic acid of various molarities,pH's, NaCl content, Citric acid of various molarities, pH's, NaClcontent, HEPES of various molarities, pH's, NaCl content, MES of variousmolarities, pH's, NaCl content, MOPS of various molarities, pH's, NaClcontent, PIPES of various molarities, pH's, NaCl content, Lactic acid ofvarious molarities, pH's, NaCl content, Phosphate of various molarities,pH's, NaCl content, Tricine of various molarities pH's, NaCl content.

[0150] Following tryptic digestion, additional processing may be carriedout, for example:

[0151] Utilizing a type of micro-chromatographic column called aC18-ZIPTIP available from the Millipore company, the followingpreparatory steps were conducted.

[0152] 1. Dilute sera in sample buffer

[0153] 2. Aspirate and dispense ZIPTIP in 50% Acetonitrile

[0154] 3. Aspirate and dispense ZIPTIP in Equilibration solution

[0155] 4. Aspirate and dispense in serum sample

[0156] 5. Aspirate and dispense ZIPTIP in Wash solution

[0157] 6. Aspirate and dispense ZIPTIP in Elution Solution

[0158] Illustrative of the various buffering compositions useful in thepresent invention are:

[0159] Sample Buffers (various low pH's): Hydrochloric acid (HCl),Formic acid, Trifluoroacetic acid (TFA), Equilibration Buffers (variouslow pH's): HCl, Formic acid, TFA;

[0160] Wash Buffers (various low pH's): HCl, Formic acid, TFA; ElutionSolutions (various low pH's and % Solvents): HCl, Formic acid, TFA;

[0161] Solvents: Ethanol, Methanol, Acetonitrile.

[0162] Spotting was then performed, for example upon a Gold Chip in thefollowing manner:

[0163] 1. Spot 2 ul of sample onto each spot

[0164] 2. Let sample partially dry

[0165] As a result of these procedures, the disease specific markersnamely proapo-A-I-protein having a molecular weight of about 1301.61daltons and a sequence of THLAPYSDELR, apolipoprotein A-I precursorhaving a molecular weight of about 1188.58 daltons and a sequence ofNLEKETEGLR, prealbumin/transthyrtin pre-albumin amyloidosis type Ihaving a molecular weight of about 1366.7595 and a sequence of(R)GSPAINVAVHVFR(K) and a molecular weight of about 2450 and a sequenceof ALGISPFHEHAEVVFTANDSGPR, fibrinogen gamma chain having a molecularweight of about 1683 daltons and a sequence of IHLISTQSAIPYALR, relatedto insulin resistance was found.

[0166]FIG. 1 is a photograph of a gel which is indicative of thepresence/absence of the marker in disease vs. control and, in caseswhere the marker is always present, the relative strength, e.g. the upor down regulation of the marker relative to categorization of diseasestate is deduced.

[0167] A method for evidencing and categorizing at least one diseasestate is disclosed. The steps taken include obtaining a sample from apatient, preferably human, and conducting MS analysis on the sample. Asa result, at least one biopolymer marker sequence or analyte thereof isisolated from the sample which undergoes evidencing and categorizing andis compared to the biopolymer marker sequence as disclosed in thepresent invention. The step of evidencing and categorizing isparticularly directed to biopolymer markers or analytes thereof linkedto at least one risk of disease development of the patient or related tothe existence of a particular disease state.

[0168] In addition, various kits are contemplated for use by the presentinvention. One such kit provides for determining the presence of thedisease specific biopolymer marker. At least one biochemical material isincorporated which is capable of specifically binding with a biomoleculewhich includes at least the disease specific biopolymer marker oranalyte thereof, and a means for determining binding between thebiochemical material and the biomolecule. The biochemical material forany of the contemplated kits, by way of example an antibody or at leastone monoclonal antibody specific therefore, or biomolecule may beimmobilized on a solid support and include at least one labeledbiochemical material which is preferably an antibody. The sampleutilized for any of the kits may be a fractionated or unfractionatedbody fluid or a tissue sample. Non-limiting examples of such fluids areblood, blood products, urine, saliva, cerebrospinal fluid, and lymph.

[0169] Further contemplated is a kit for diagnosing, determiningrisk-assessment, and identifying therapeutic avenues related to adisease state. This kit includes at least one biochemical material whichis capable of specifically binding with a biomolecule which includes atleast one biopolymer marker including the sequence of the particulardisease specific biopolymer marker or an analyte thereof related to thedisease state. Also included is a means for determining binding betweenthe biochemical material and the biomolecule, whereby at least oneanalysis to determine a presence of a marker, analyte thereof, or abiochemical material specific thereto, is carried out on a sample. Aspreviously described, analysis may be carried out on a single sample ormultiple samples.

[0170] In accordance with various stated objectives of the invention,the skilled artisan, in possession of the specific disease specificmarker as instantly disclosed, would readily carry out known techniquesin order to raise purified biochemical materials, e.g. monoclonal and/orpolyclonal antibodies, which are useful in the production of methods anddevices useful as point-of-care rapid assay diagnostic or riskassessment devices as are known in the art.

[0171] The specific disease markers which are analyzed according to themethod of the invention are released into the circulation and may bepresent in the blood or in any blood product, for example plasma, serum,cytolyzed blood, e.g. by treatment with hypotonic buffer or detergentsand dilutions and preparations thereof, and other body fluids, e.g. CSF,saliva, urine, lymph, and the like. The presence of each marker isdetermined using antibodies specific for each of the markers anddetecting specific binding of each antibody to its respective marker.Any suitable direct or indirect assay method may be used to determinethe level of each of the specific markers measured according to theinvention. The assays may be competitive assays, sandwich assays, andthe label may be selected from the group of well-known labels such asradioimmunoassay, fluorescent or chemiluminescence immunoassay, orimmunoPCR technology. Extensive discussion of the known immunoassaytechniques is not required here since these are known to those ofskilled in the art. See Takahashi et al. (Clin Chem 1999;45(8):1307) fora detailed example of an assay.

[0172] A monoclonal antibody specific against the disease markersequence isolated by the present invention may be produced, for example,by the polyethylene glycol (PEG) mediated cell fusion method, in amanner well-known in the art.

[0173] Traditionally, monoclonal antibodies have been made according tofundamental principles laid down by Kohler and Milstein. Mice areimmunized with antigens, with or without, adjuvants. The splenocytes areharvested from the spleen for fusion with immortalized hybridomapartners. These are seeded into microtiter plates where they can secreteantibodies into the supernatant that is used for cell culture. To selectfrom the hybridomas that have been plated for the ones that produceantibodies of interest, the hybridoma supernatants are usually testedfor antibody binding to antigens in an ELISA (enzyme linkedimmunosorbent assay) assay. The idea is that the wells that contain thehybridoma of interest will contain antibodies that will bind most avidlyto the test antigen, usually the immunizing antigen. These wells arethen subcloned in limiting dilution fashion to produce monoclonalhybridomas. The selection for the clones of interest is repeated usingan ELISA assay to test for antibody binding. Therefore, the principlethat has been propagated is that in the production of monoclonalantibodies the hybridomas that produce the most avidly bindingantibodies are the ones that are selected from among all the hybridomasthat were initially produced. That is to say, the preferred antibody isthe one with highest affinity for the antigen of interest.

[0174] There have been many modifications of this procedure such asusing whole cells for immunization. In this method, instead of usingpurified antigens, entire cells are used for immunization. Anothermodification is the use of cellular ELISA for screening. In this methodinstead of using purified antigens as the target in the ELISA, fixedcells are used. In addition to ELISA tests, complement mediatedcytotoxicity assays have also been used in the screening process.However, antibody-binding assays were used in conjunction withcytotoxicity tests. Thus, despite many modifications, the process ofproducing monoclonal antibodies relies on antibody binding to the testantigen as an endpoint.

[0175] The purified monoclonal antibody is utilized for immunochemicalstudies.

[0176] Polyclonal antibody production and purification utilizing one ormore animal hosts in a manner well-known in the art can be performed bya skilled artisan.

[0177] Another objective of the present invention is to provide reagentsfor use in diagnostic assays for the detection of the particularlyisolated disease specific marker sequences of the present invention.

[0178] In one mode of this embodiment, the marker sequences of thepresent invention may be used as antigens in immunoassays for thedetection of those individuals suffering from the disease known to beevidenced by said marker sequence. Such assays may include but are notlimited to: radioimmunoassay, enzyme-linked immunosorbent assay (ELISA),“sandwich” assays, precipitin reactions, gel diffusion immunodiffusionassay, agglutination assay, fluorescent immunoassays, protein A or Gimmunoassays and immunoelectrophoresis assays.

[0179] According to the present invention, monoclonal or polyclonalantibodies produced against the disease specific marker sequence of theinstant invention are useful in an immunoassay on samples of blood orblood products such as serum, plasma or the like, cerebrospinal fluid orother body fluid, e.g. saliva, urine, lymph, and the like, to diagnosepatients with the characteristic disease state linked to said markersequence. The antibodies can be used in any type of immunoassay. Thisincludes both the two-site sandwich assay and the single siteimmunoassay of the non-competitive type, as well as in traditionalcompetitive binding assays.

[0180] Particularly preferred, for ease and simplicity of detection, andits quantitative nature, is the sandwich or double antibody assay ofwhich a number of variations exist, all of which are contemplated by thepresent invention. For example, in a typical sandwich assay, unlabeledantibody is immobilized on a solid phase, e.g. microtiter plate, and thesample to be tested is added. After a certain period of incubation toallow formation of an antibody-antigen complex, a second antibody,labeled with a reporter molecule capable of inducing a detectablesignal, is added and incubation is continued to allow sufficient timefor binding with the antigen at a different site, resulting with aformation of a complex of antibody-antigen-labeled antibody. Thepresence of the antigen is determined by observation of a signal whichmay be quantitated by comparison with control samples containing knownamounts of antigen.

[0181] Antibodies may also be utilized against the disease specificmarkers, as haptens, to create an antibody response against the proteinto which it binds, thereby identifying targets for treatment of thedisease or a sub-class thereof.

[0182] Lastly, the markers and associated antibodies provide a tool formonitoring the progress of a patient during a therapeutic treatment, soas to determine the usefulness of a novel therapeutic agent.

[0183] All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

[0184] It is to be understood that while a certain form of the inventionis illustrated, it is not to be limited to the specific form orarrangement herein described and shown. It will be apparent to thoseskilled in the art that various changes may be made without departingfrom the scope of the invention and the invention is not to beconsidered limited to what is shown and described in the specificationand drawings/figures.

[0185] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objectives and obtain theends and advantages mentioned, as well as those inherent therein. Theoligonucleotides, peptides, polypeptides, biologically relatedcompounds, methods, procedures and techniques described herein arepresently representative of the preferred embodiments, are intended tobe exemplary and are not intended as limitations on the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the appended claims. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

What is claimed is:
 1. A biopolymer marker selected from the groupconsisting of sequence ID THLAPYSDELR, NLEKETEGLR, (R)GSPAINVAVHVFR(K),ALGISPFHEHAEVVFTANDSGPR, IHLISTQSAIPYALR or at least one analyte thereofuseful in indicating at least one particular disease state.
 2. Thebiopolymer marker of claim 1 wherein said disease state is predictive ofAlzheimers disease.
 3. A method for evidencing and categorizing at leastone disease state comprising: obtaining a sample from a patient;conducting mass spectrometric analysis on said sample; evidencing andcategorizing at least one biopolymer marker sequence or analyte thereofisolated from said sample; and, comparing said at least one isolatedbiopolymer marker sequence or analyte thereof to the biopolymer markersequence as set forth in claim 1; wherein correlation of said isolatedbiopolymer marker and said biopolymer marker sequence as set forth inclaim 1 evidences and categorizes said at least one disease state. 4.The method of claim 3, wherein said step of evidencing and categorizingis particularly directed to biopolymer markers or analytes thereoflinked to at least one risk of disease development of said patient. 5.The method of claim 3, wherein said step of evidencing and categorizingis particularly directed to biopolymer markers or analytes thereofrelated to the existence of a particular disease state.
 6. The method ofclaim 3, wherein the sample is an unfractionated body fluid or a tissuesample.
 7. The method of claim 3, wherein said sample is at least one ofthe group consisting of blood, blood products, urine, saliva,cerebrospinal fluid, and lymph.
 8. The method of claim 3, wherein saidmass spectrometric analysis is selected from the group consisting ofSurface Enhanced Laser Desorption Ionization (SELDI) mass spectrometry(MS), Maldi Qq TOF, MS/MS, TOF-TOF, and ESI-Q-TOF or an ION-TRAP.
 9. Themethod of claim 3, wherein said patient is a human.
 10. A diagnosticassay kit for determining the presence of the biopolymer marker oranalyte thereof of claim 1 comprising: at least one biochemical materialwhich is capable of specifically binding with a biomolecule whichincludes at least said biopolymer marker or analyte thereof, and meansfor determining binding between said biochemical material and saidbiomolecule; whereby at least one analysis to determine a presence of amarker, analyte thereof, or a biochemical material specific thereto, iscarried out on a sample.
 11. The diagnostic assay kit of claim 10,wherein said biochemical material or biomolecule is immobilized on asolid support.
 12. The diagnostic assay kit of claim 10 including: atleast one labeled biochemical material.
 13. The diagnostic assay kit ofclaim 10, wherein said biochemical material is an antibody.
 14. Thediagnostic assay kit of claim 12, wherein said labeled biochemicalmaterial is an antibody.
 15. The diagnostic assay kit of claim 10,wherein the sample is an unfractionated body fluid or a tissue sample.16. The diagnostic assay kit of claim 10, wherein said sample is atleast one of the group consisting of blood, blood products, urine,saliva, cerebrospinal fluid, and lymph.
 17. The diagnostic assay kit ofclaim 10, wherein said biochemical material is at least one monoclonalantibody specific therefore.
 18. A kit for diagnosing, determiningrisk-assessment, and identifying therapeutic avenues related to adisease state comprising: at least one biochemical material which iscapable of specifically binding with a biomolecule which includes atleast one biopolymer marker selected from the group consisting ofsequence ID THLAPYSDELR, NLEKETEGLR,(R)GSPAINVAVHVFR(K),ALGISPFHEHAEVVFTANDSGPR, IHLISTQSAIPYALR or analytethereof related to said disease state; and means for determining bindingbetween said biochemical material and said biomolecule; whereby at leastone analysis to determine a presence of a marker, analyte thereof, or abiochemical material specific thereto, is carried out on a sample. 19.The kit of claim 18, wherein said biochemical material or biomolecule isimmobilized on a solid support.
 20. The kit of claim 18 including: atleast one labeled biochemical material.
 21. The kit of claim 18, whereinsaid biochemical material is an antibody.
 22. The kit of claim 20,wherein said labeled biochemical material is an antibody.
 23. The kit ofclaim 18, wherein the sample is an unfractionated body fluid or a tissuesample.
 24. The kit of claim 18, wherein said sample is at least one ofthe group consisting of blood, blood products, urine, saliva,cerebrospinal fluid, and lymph.
 25. The kit of claim 18, wherein saidbiochemical material is at least one monoclonal antibody specifictherefore.
 26. The kit of claim 18, wherein said diagnosing, determiningrisk assessment, and identifying therapeutic avenues is carried out on asingle sample.
 27. The kit of claim 18, wherein said diagnosing,determining risk assessment, and identifying therapeutic avenues iscarried out on multiple samples such that at least one analysis iscarried out on a first sample and at least another analysis is carriedout on a second sample.
 28. The kit of claim 27, wherein said first andsecond samples are obtained at different time periods.
 29. Polyclonalantibodies produced against a marker sequence ID selected from the groupconsisting of sequence THLAPYSDELR, NLEKETEGLR, (R)GSPAINVAVHVFR(K),ALGISPFHEHAEVVFTANDSGPR, IHLISTQSAIPYALR or at least one analyte thereofin at least one animal host.
 30. An antibody that specifically binds abiopolymer including a marker selected from the group consisting ofsequence ID THLAPYSDELR, NLEKETEGLR, (R)GSPAINVAVHVFR(K),ALGISPFHEHAEVVFTANDSGPR, IHLISTQSAIPYALR or at least one analytethereof.
 31. The antibody of claim 30 that is a monoclonal antibody. 32.The antibody of claim 30 that is a polyclonal antibody.
 33. A processfor identifying therapeutic avenues related to a disease statecomprising: conducting an analysis as provided by the kit of claim 18;and interacting with a biopolymer selected from the group consisting ofsequence ID THLAPYSDELR, NLEKETEGLR,(R)GSPAINVAVHVFR(K),ALGISPFHEHAEVVFTANDSGPR, IHLISTQSAIPYALR or at leastone analyte thereof; whereby therapeutic avenues are developed.
 34. Theprocess for identifying therapeutic avenues related to a disease statein accordance with claim 33, wherein said therapeutic avenues regulatethe presence or absence of the biopolymer selected from the groupconsisting of sequence ID THLAPYSDELR, NLEKETEGLR,(R)GSPAINVAVHVFR(K),ALGISPFHEHAEVVFTANDSGPR, IHLISTQSAIPYALR or at leastone analyte thereof.
 35. The process for identifying therapeutic avenuesrelated to a disease state in accordance with claim 33, wherein saidtherapeutic avenues developed include at least one avenue selected froma group consisting of 1)utilization and recognition of said biopolymermarkers, variants or moieties thereof as direct therapeutic modalities,either alone or in conjunction with an effective amount of apharmaceutically effective carrier; 2)validation of therapeuticmodalities or disease preventative agents as a function of biopolymermarker presence or concentration; 3)treatment or prevention of a diseasestate by formation of disease intervention modalities; 4)use ofbiopolymer markers or moieties thereof as a means of elucidatingtherapeutically viable agents, 5)instigation of a therapeuticimmunological response; and 6) synthesis of molecular structures relatedto said biopolymer markers, moieties or variants thereof which areconstructed and arranged to therapeutically intervene in said diseasestate.
 36. The process for identifying therapeutic avenues related to adisease state in accordance with claim 35, wherein said treatment orprevention of a disease state by formation of disease interventionmodalities is the formation of biopolymer/ligand conjugates whichintervene at receptor sites to prevent, delay or reverse a diseaseprocess.
 37. The process for identifying therapeutic avenues related toa disease state in accordance with claim 35, wherein said means ofelucidating therapeutically viable agents includes use of abacteriophage peptide display library or a bacteriophage antibodylibrary.
 38. A process for regulating a disease state by controlling thepresence or absence of a biopolymer selected from the group consistingof sequence ID THLAPYSDELR, NLEKETEGLR, (R)GSPAINVAVHVFR(K),ALGISPFHEHAEVVFTANDSGPR, IHLISTQSAIPYALR or at least one analytethereof.